title: Biochemical activities of Streptococcus pneumoniae serotype 2 capsular glycosyltransferases and significance of suppressor mutations affecting the initiating glycosyltransferase Cps2E.
abstract: The capsular polysaccharide (CPS) is essential for Streptococcus pneumoniae virulence. Its synthesis requires multiple enzymes, and defects that block completion of the pathway can be lethal in the absence of secondary suppressor mutations. In this study, we examined the functions of three capsular glycosyltransferases (Cps2F, Cps2G, and Cps2I) involved in serotype 2 CPS synthesis, whose deletions select for secondary mutations. We demonstrate that Cps2F is a rhamnosyltransferase that catalyzes addition of the third and fourth sugars in the capsule repeat unit, while Cps2G adds the fifth sugar (glucose). Addition of the terminal residue (glucuronic acid) could not be detected; however, activities of the other glycosyltransferases together with bioinformatic analyses suggest that this step is mediated by Cps2I. Most of the secondary suppressor mutations resulting from loss of these enzymes occur in cps2E, the gene encoding the initiating glycosyltransferase. Examination of the 69 S. pneumoniae serotypes containing Cps2E homologues yielded a consensus amino acid sequence for this protein and demonstrated that there is a highly significant association between the residues that are 100% conserved and those altered by suppressor mutations. Cps2E contains an extracytoplasmic loop whose function is unknown. Among our collection of mutants, six contained missense mutations affecting amino acids in the extracytoplasmic loop. These residues are highly conserved among S. pneumoniae Cps2E homologues, and mutations therein severely reduced CPS synthesis and Cps2E activity. The critical functions of these amino acids suggest a role for the Cps2E extracytoplasmic loop in initiation, and possibly regulation, of capsule synthesis.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: David B A
authors.initials: DB
authors.lastname: James
authors.forename: Kanupriya
authors.initials: K
authors.lastname: Gupta
authors.forename: Jocelyn R
authors.initials: JR
authors.lastname: Hauser
authors.forename: Janet
authors.initials: J
authors.lastname: Yother
mesh_terms: D016667:Bacterial Capsules / Q000378:metabolism*; D017353:Gene Deletion; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic
chemical_list: D016695:Glycosyltransferases